7. Project Summary/Abstract Adult human skin heals by developing fibrotic scar tissue, which can result in devastating disfigurement, growth restriction, and permanent functional loss. Despite a plethora of clinical options, no current treatment strategies successfully prevent or reverse this fibrotic process, and scars and their sequelae cost the United States over $20 billion every year. Progress towards the development of new therapies has been significantly hindered by a lack of understanding of the specific cell populations responsible for scarring. In 2015, our group reported that Engrailed-1 (En-1) lineage-positive fibroblasts (EPFs) are responsible for the vast majority of dorsal scar production in postnatal mice. In early fetal gestation, mice heal scarlessly via skin regeneration, an ideal outcome mediated by En-1 lineage-negative fibroblasts (ENFs; the predominant fetal fibroblast). However, it has not been established if ENFs contribute to postnatal wound healing. In this proposal, we explore for the first time the postnatal conversion of ENFs to pro-fibrotic EPFs (postnatally-derived EPFs; pEPFs) within the wound environment. First, histology, immunohistochemistry, and wounding in a novel transgenic mouse model will be used to study the conversion of ENFs to pEPFs during wound healing. By examining the behavior of ENF subpopulations (derived from papillary dermis, reticular dermis, and hypodermis) in the wound environment and confirming our findings in a tamoxifen-inducible mouse model of En-1 activation, we will precisely define the ENF population that gives rise to pro-fibrotic pEPFs. Second, we will establish the specific wound environment cues that drive ENF-to-EPF transition. Given that mechanical forces are known to modulate both scar burden and fibroblast activity, we will use in vitro and in vivo models to examine the effects of mechanical environment on En-1 activation. We will further use transcriptomic and epigenomic profiling to explore the role of mechanotransduction signaling in ENF-to-EPF transition and pEPF function. Third, having established a mechanotransduction mechanism underlying En-1 activation in wound ENFs, we will inhibit mechanotransduction signaling with the goal of blocking ENF-to-EPF transition. Specifically, we will assess whether blocking mechanotransduction results in ENF-mediated wound healing with reduced fibrosis. Our ultimate translational goal is to develop therapeutics that target fibrogenic fibroblasts to promote regenerative healing. Collectively, the proposed work will significantly enhance our understanding of the key molecular and cellular determinants of cutaneous scarring, inform the development of novel anti-scarring therapies, and shed light on the cellular origin of dermal scarring fibroblasts.